Human Blood Cultures Research Articles

Sulfur dioxide-induced lymphocyte defects in human peripheral blood cultures

Human lymphocyte cultures were prepared from peripheral blood of healthy males by a standard technique utilizing phytohemagglutinin. Nine cultures were established in each of five experiments. One, designated control, was allowed to grow without interruption for 3 days. Each experimental culture received a single exposure to 100 cc of 5.7 ppm SO 2 in air by bubbling the gas through the culture medium at either 0, 1, 2, or 3 days of incubation. An additional four cultures received an identical amount of the same quality of air minus the SO 2 at each time interval. All cultures were harvested as smears and hypotonic spread preparations at 72 hours after treatment for 1 hour with colchicine and 3H-thymidine. Statistical analysis revealed significantly reduced cell growth (enlargement), DNA synthesis, and mitotic indices in the cultures exposed to sulfur dioxide at initiation of incubation compared to controls and air-treated cultures. In addition, chromosome abnormalities, mainly in the form of clumping, were observed in the SO 2-treated cultures. Lymphocyte damage as caused by sulfur dioxide exposure is offered as one explanation for the reduced immunological responses observed in laboratory animals after inhalation of the gas in vivo.

Chromosome Aberrations Induced by an Ultrasonic Fetal Pulse Detector

Human blood cultures were exposed to ultrasound from an ultrasonic fetal heart detector for periods of one and two hours. Considerable increases in the number of chromosome aberrations over control values were found.

Phenothiazines and Chromosome Damage

A higher incidence of chromatid breakage was observed in human peripheral blood cultures derived from three individuals under phenothiazine treatment as compared to those derived from four normal, healthy individuals. The phenothiazines involved in the study are chlorpromazine (thorazine) and trifluoperazine hydrochloride (stelazine). The mean per cent breaks in the cultures derived from treated individuals was 27.2±6.68 while in cultures derived from control individuals, it was 10.2±1.54 indicating that either or both phenothiazine drugs may affect the cell in vivo in an adverse manner. In the course of this study, it became necessary to develop a somewhat detailed method for the identification of chromatid breaks.

CHROMOSOME POLYPLOIDIZATION IN HUMAN LEUKOCYTES INDUCED BY RADIATION

A great quantity of literature has accumulated on the effects of radiation and radiomimetic agents upon cells. Much of this work has been performed on mammalian cells both in vivo and in tissue cultures. Some of these damaging effects can be seen in dividing cells. After the development of a technique for tissue culture of human peripheral blood, we were able to demonstrate abnormal patterns and chromosomal polyploidy in cultures from patients who had been treated with A-8103 (a piperazine derivative with antitumor activity) , l nitrogen mustard, 6-mercaptopurine, and A-649 (another antitumor agent).2 During treatment and for some time after treatment in these patients, structural changes could be seen in the chromosomes as either chromatid or chromosome deletions and chromosomal breakage. Abnormally shaped chromosomes were also seen, particularly ring forms and dicentrics. Although aneuploidy was observed, the major numerical variation consisted of metaphases showing polyploidy with random distribution of chromosomes and diplochromosomes. When normal human leukocytes were tested in vitro with the chemotherapeutic agents already mentioned, as well as with streptonigrin and cycl~phosphamide,~ similar results were obtained. From these studies we postulated that two different types of damage were induced in the cells by the chemicals. One type of damage is apparent in the hereditary material, and it can be characterized by the structural chromosomal changes. The second type of damage is interpreted as damage to the mitotic apparatus of the cells, as evidenced by the induction of polyploidy. Simultaneously, initial studies from patients treated with x-rays and cobalt-60 demonstrated that the same type of chromosomal anomalies were present in their peripheral leukocytes as those induced with chemotherapeutic agents. Generally, these were structural variations and chromosome polyploidization. Certainly, chromosomal changes have been described in association with x r a d i a t i ~ n ~ , ~ and 1311 therapye,' and, rarely, following diagnostic x-radiation. However, in most of these reports, the occurrence of polyploidy was overlooked, and when polyploidy was mentioned, it was not characterized. Since both structural damage as well as polyploidy lead to genetic transformation or to somatic cell death, we thought it of paramount interest to determine whether or not induction of chromosomal polyploidy by radiation was a constant feature in human somatic cells. Such effects may have a bearing on both the induction as well as the treatment of cancer. For the present study, we determined not only polyploidy, but also the amount of aneuploidy, chromosomal breakage, ring forms, translocations, dicentrics, and tricentrics as well as other rearrangements appearing in patients exposed to radiation therapy. In addition, normal human leukocytes were exposed to x-rays in cultures, and information of induced damage was obtained.

Mouse germ cells and LSD-25.

Acute and chronic studies of male and female meiosis in an Imperial-Chemical-Industries-derived strain of mice were carried out with LSD-25. Over a wide range of doses claimed to be effective in teratogenesis and male meiosis in the mouse and in the production of chromosome damage in leucocyte cultures of human blood, no convincing effect was detected on the chromosomes of first or second meiotic divisions, nor in those of the bone-marrow or of the few spermatogonial metaphases that were studied.

Opossum lymphocytes in vitro: a valuable tool for cytogenetic investigations.

Lymphocytes obtained from cardiac blood and thoracic duct lymph of the opossum (<i>Didelphis virginiana</i>) were incubated in short-term culture with phytohemagglutinin. Relatively pure inocula of lymphocytes were prepared through elimination of erythrocytes and granulocytes. A critical condition for successful culture growth was found to be the use of heat-inactivated rat serum in place of autologous or isologous sera. Because of rapidly occurring bursts of DNA synthesis, cellular transformation and mitotic activity, cultures were harvested at one half the incubation time common to human blood cultures and those of other laboratory animals. Opossum lymphocytes provide excellent material for cytogenetic investigations because of this short incubation requirement and because their chromosomes are large and few in number. Particularly significant is the ease with which the sex chromosomes may be identified.

An electron-microscope study of the in vitro transformation of human leucocytes. II. Transformation to macrophages.

Abstract Macrophages which develop in cultures of human peripheral blood have the typical ultrastructural features of macrophages occurring in other situations, in vivo and in vitro. The cultured cells usually have irregular nuclei and the cytoplasm possesses numerous digestion vacuoles containing debris; the cell outline is irregular with many filopodia. Other cells were laden with membrane-bounded granular bodies containing periodic-structured material of regular outline. Although it is concluded that no evidence about cell ancestry can be adduced from this morphological study, it seems likely from other work that many of these macrophages arise from lymphocytes.

Autoradiographical study on the time of nuclear protein synthesis in human leucocyte blood culture

An autoradiographic study on the time of nuclear protein synthesis was carried out on human lymphocytes cultured in vitro. Experiments with pulse and prolonged labeling with 3H-arg and 3H-lys allow the conclusion that the synthesis of proteins and chromosomal proteins in particular proceed during all phases of mitotic cycle. Protein synthesis takes place in metaphase and prophase. The rate of protein synthesis in G1-phase is about 2–3 times less than that in S-G2-phases.

Sporadic triploid cells in human blood and fibroblast cultures.

Sporadic triploid cells have been found in two patients with otherwise normal karyotypes and in one normal placenta. Sporadic triploid cells are probably not very rare. Further data should be collected in order to elucidate the interesting problem of the origin of triploid cells.

Method for demonstrating the chromosomes in adult human spleen cells.

To obtain chromosome preparations from in vitro cultures of human peripheral blood is now a routine laboratory procedure. Baker and Atkins1 and Bain and Gauld2 have extended this technique to human foetal and infant spleen and adult lymph nodes. The present communication describes the further extension of this method, to demonstrate the chromosomes of adult human spleen cells following culture in vitro.

RNA Synthesis in Cultures of Normal Human Peripheral Blood

Abstract RNA and DNA synthesis were measured in cultures of normal human peripheral blood using tritiated cytidine and thymidine and autoradiographic technics. RNA synthesis preceded DNA synthesis by 24 hours. RNA synthesis occurred predominantly in the large and medium-sized "blast-like" cells, but did occur, to a lesser extent, in the small lymphocytes. RNA synthesis did not occur in the absence of phytohemagglutinin, nor did DNA synthesis. Mechanisms of action of phytohemagglutinin are discussed with particular reference to its possible antigenic nature.

Transformation of Lymphocytes in Cultures of Human Peripheral Blood

AbstractPrevious tracer studies have demonstrated the lymphocytic origin of proliferating cells in blood cultures. Detailed morphologic observations are reported on the transformation of small lymphocytes into larger cells capable of division in tissue culture of human peripheral blood. The large cells have ample cytoplasm with multivesicular bodies, well-developed Golgi apparatus, scanty endoplasmic reticulum, ample ribosomes, and variable fat-laden vacuoles as well as peculiar granular inclusions, large and occasionally bizarre nuclei with prominent nucleoli. Under the condition of culture no further differentiation of these cells has occurred.The potentialities of the small lymphocyte are discussed in the light of recent radiobiologic and cytogenetic investigations.

Induction of leucocyte growth in cultures of human peripheral blood.

Induction of leucocyte growth in cultures of human peripheral blood.

The Kinetics of Cell Proliferation in Cultures of Human Peripheral Blood

Abstract The kinetics of growth of peripheral blood cells in tissue culture are described. There was a rapid increase of cells in DNA synthesis beginning at 24 hours and by 72 hours 40-45 per cent of the cells were in DNA synthesis. Mitotic rates of ∼ 1 per cent/hour were seen after 72 hours in cultures. Studies with tritiated thymidine and colchicine indicated that these cells represent transformed lymphocytes and do not arise from the cells in DNA synthesis at time zero or from any other small population of cells.

Glass substrate cultures of human blood and bone marrow cells

Methods and results of culturing human blood and bone marrow cells on glass substrates are reported. A pattern of culture divisible into three successive but overlapping phases was observed. Cells of the first phase, recognisable as normal blood cells, are more suitably studied by other techniques. The large granular cells of the second phase and the fibroblast-like cells of the third phase are described and their origin discussed. Glass substrate cultures of bone marrow cells are a convenient and reliable source of human fibroblast-like cells for morphological, physiological and pharmocological experiments. They have no value as an aid to diagnosis.

Cytological investigations on cultures of normal human blood

1. In the cultures of leucocytes of normal human blood under definite conditions of cultivation there appear periods of mass multiplication of cells in mitotic fashion. 2. The mass multiplication is observed in the polyblasts (macrophages) at the stage of their mutation into stable cells, and in the fibrocytes originating in haemocultures from polyblasts. 3. For obtaining the maximal quantity of mitoses the best time for fixation is the second 24 hours after the renewal of the medium, this latter being performed not later than the end of the fourth 24 hours after the planting of the culture. 4. After appropriate fixation, the figures of divisions may be used for karyological analysis. 5. The method proposed enlarges the possibilities of the investigation of the human karyotype, and enables us to undertake the karyological investigation of any anthropogenetical or medical phenomena.

Variations in Monocytic Response to Peroxydase Test.

Naegeli states that almost all, if not all, of the human monocytes are peroxydase reacting. The author found no peroxydase-reacting monocytes in the blood of a rabbit. In a normal adult man there were present 3.4% peroxydase mononuclear phagocytes and 1.1% phagocytes that did not react to the peroxydase test. The conclusion was that the peroxydase cells of human blood are of myeloid origin, which is the view held by Naegeli. The negative phagocytes were thought to be the true monocytes derived from the reticular portion of lymphoid tissue. Recently some unpublished observations have brought me to a consideration of the alternative view, namely, that the peroxydase-positive phagocyte gets its granulation secondarily in a more or less accidental manner. The conclusions arrived at in this paper are based on tissue cultures of blood leukocytes. Methods and Results: Numerous cultures of human blood were made by the coverglass method and examined by the peroxydase test daily from one day to 4 weeks. This was done on the assumption that the peroxydase granulation of the monocytes would disappear, provided the granulation was acquired secondarily and was not an essential part of the cell cytoplasm. After one month in the incubator large peroxydase reacting phagocytes were present in the cultures. Never was the number of phagocytes great and no conclusion was possible in regard to the proportion of positive and negative cells. Rhoads and Parker found at the end of 2 weeks of incubation both positive and negative mononuclear phagocytes in normal human blood. The next procedure was to culture rabbit leukocytes in contact with variable amounts of human blood: Rabbit blood was cultured on sterile cover-glasses covered with smears of human blood made with aseptic precautions. The smears were kept dry in the air for 2 hours. The rabbit blood was obtained from the heart and drops of blood placed directly on the covers from the small caliber needle. The covers which had previously been rimmed with sterile vaseline were inverted over the well and placed in the incubator within 5 minutes after withdrawal of the blood from the heart. After about one hour the covers were sealed permanently with paraffin. At the end of 2 weeks as many as 500 large mononucleated cells were present in some of the cultures while in the human blood cultures mentioned above the cells at the most numbered a few dozen.